The field of the present invention relates to methods of expressing relatively high levels of recombinant tobacco osmotin in a microorganism. The recombinant tobacco osmotin thereby expressed may be purified and utilized as a biocidal agent or a therapeutic agent in medical applications.
Pathogenesis-related proteins (PR) are produced by plants in response to environmental stress, such as extremes of salinity, and pathogen infections (Kononowicz et al. 1992). Because of the nature and purpose of host plants, there is a great potential for some PR proteins to be used in controlling food borne pathogens. Among PR proteins, osmotin is one of the best studied which has shown biocidal activity (Narasimhan et al. 2005; Narasimhan et al. 2001). Because the biocidal mechanisms of osmotin are somewhat hormonal, theoretically, osmotin should be more potent than enzymatic and enzyme-inhibitory biocides. Moreover, osmotin is safe for human intake because of its existence in many plants, meaning that humans frequently consume osmotin in diets of vegetables, fruit, or nuts.
Recent studies of the fungicidal mechanisms of tobacco osmotin (OSM) have shown that OSM binds to yeast surface protein, PHO36, and mediates the signal cascades leading to apoptosis. Because PH036 is homologous to human adiponectin receptor, OSM has been proposed to be an analogue of adiponectin carrying similar functions in human (Narasimhan et al., 2005). Despite the early discovery and characterization of OSM (Singh et al., 1987) and its potential applications in treating fungal infections, diabetes, and obesity in humans (Min et al., 2004; Narasimhan et al., 2005) and in controlling food borne pathogens, osmotin has never been widely investigated or applied because of its limited availability. Osmotin has proved to be refractory to common systems for overexpression. Currently, the only source or OSM is a purified form from salt-adapted tobacco cell culture of Wisconsin 38 (Singh et al., 1987).
A transgenic plant that includes OSM driven by a strong promoter, such as cauliflower mosaic virus, theoretically should be a good source for OSM. Liu et al. (1994) successfully obtained OSM overexpressed potato by Agrobacterium-mediated transformation. However, no one has reported the production of OSM from a transgenic plant. OSM has been suggested to form multimers (up to 20 mers, Singh, personal communication). This makes OSM extraction from plant tissues and elution in chromatographic purification difficult if not impossible. Therefore, scientists have been seeking more facile expression systems, such as E. coli. However, OSM is somewhat toxic to bacteria, resulting in a very low yield in bacterial expression systems. For these reasons, better expression systems for OSM are desirable.